The diagnosis and management of glaucoma requires sensitive methods for detecting and measuring damage to retinal nerve fibers. Newly-developed quantitative methods for optically assessing the retinal nerve fiber layer (RNFL) promise to enhance sensitivity and objectivity, but more basic knowledge is needed to interpret and improve them. The long-term objectives of this research are to provide a comprehensive quantitative description of the optical properties of the RNFL, and to establish the anatomical basis for its reflectance. Experiments with toad retina showed that the RNFL reflectance arises from uniformly-distributed cylindrical structures. A new theoretical model based on the ultrastructure of nerve fiber bundles suggests that axonal membranes dominate the reflectance, but preliminary experiments suggest that a substantial fraction of the RNFL reflectance comes from microtubules. The model predicts that membrane and microtubule mechanisms should behave differently in polarized light. The specific aims of this project are to: 1) perform reflectometry of rat RNFL to confirm that properties discovered in toad occur more generally, and to test the prediction that axonal membranes dominate the RNFL reflectance; 2) use colchicine to depolymerize microtubules in order to test the hypothesis that the RNFL reflectance arises from light scattered by microtubules; 3) use polarized light to broaden the quantitative description of the RNFL reflectance and to assess the relative contributions of membranes and microtubules to the reflectance mechanisms; and 4) study quantitatively the ultrastructure of RNFL tissue from toad, rat, and human in order to relate reflectometry to anatomy. Reflectometric experiments will be performed with a unique apparatus, an imaging microreflectometer, on in vitro preparations, and morphological measurements will be obtained on the same tissue. By relating reflectometry to morphology for a variety of RNFL reflectance mechanism. Additionally the data obtained will broaden the quantitative description of the RNFL reflectance and provide a solid foundation of knowledge upon which to develop RNFL assessment methods for glaucoma.